Abstract: Flow-induced vasodilation (FIV) is a hallmark of the endothelial response to flow and an essential mechanism for the control of blood flow to the microcirculation. It is well established that a key mechanism responsible for FIV is generation of nitric oxide (NO). Our recent study discovered that FIV and flow-induced generation of NO in resistance arteries of mice and humans critically depend on endothelial inwardly-rectifying K+ channels (Kir2.1). We also established that Kir2.1 regulate endothelial NO synthase (eNOS) via a serine/threonine kinase Akt1. This was particularly interesting and important because Kir channels have long been known to be sensitive to shear stress but their role in endothelial responses to flow remained unknown. The goals of this proposal are to determine the mechanisms by which Kir2.1 channels couple hemodynamic shear stress forces to activation of endothelial NO synthase (eNOS) and NO production and to evaluate the role of endothelial Kir channels in vasoreactivity of human vessels in hypertension. Our first aim is to elucidate the mechanism responsible for the sensitivity of Kir2.1 channels to shear stress, which is currently completely unknown. Our preliminary data show that flow-sensitivity of Kir2.1 is abrogated by enzymatic degradation of Heparan Sulphate (HS)-Glycocalyx and reduced in ECs isolated from Sydecan1-/- mice. We propose, therefore, that flow-induced activation of Kir channels is mediated by the endothelial Glycocalyx, specifically Syndecan-1, and possibly other elements of HS-Glycocalyx. We also propose that Kir2.1 interacts directly with Syndecan-1, and elucidate the mechanism of this interaction. Our second aim focuses on the mechanism that couples Kir2.1 to the downstream Akt1 signaling pathway. It is well-known that flow-induced activation of AKT1 requires its translocation and recruitment to the membrane via association with a phospholipid PIP3. We propose that Kir enhances the association of Akt1 with PIP3 and thus facilitates its recruitment to the membrane, resulting in increased Akt1 phosphorylation. We also explore the possibilities that flow-induced activation of Kir2.1 may regulate the upstream events, such as activation PI3K and its recruitment to VEGFR2 mechanosensing complex or inhibit a phosphatase PTEN that converts PIP3 to PIP2. This signaling mechanism is explored in primary endothelial cells and in intact resistance arteries freshly-harvested from mice. A new endothelial-specific inducible mouse model of Kir2.1 deficiency has been generated in our lab to achieve these goals. In aim 3, we propose to test the hypothesis that microvascular endothelial Kir function is depressed during human hypertension. This aim is based on our preliminary data showing decreased contribution of Kir2.1 to FIV in a pilot cohort of hypertensive patients. In this study, we will recruit 3 groups of subjects that include patients with pre-hypertension or stage 1 hypertension and healthy controls. We will also determine whether the loss of Kir2.1 contribution to FIV should be attributed to the loss of the functional expression of Kir2.1 channels or to their impaired coupling to the downstream signaling. Finally, we will also determine whether impaired FIV in hypertensive patients may be rescued by restoring Kir2.1 activity.